Proteolytic extract from bromelain for the treatment of connective tissue disorders

ABSTRACT

The present invention relates to a proteolytic extract obtained from bromelain for the treatment of connective tissue diseases. In particular, the present invention relates to a pharmaceutical composition comprising a proteolytic extract obtained from bromelain for the treatment of diseases such as Dupuytren&#39;s disease and Peyronie&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/233,082 filed on Jan. 15, 2014, which is a 371 filing ofInternational patent application no. PCT/IL2012/050261 filed on Jul. 19,2012, which claims the benefit of U.S. provisional patent applicationNo. 61/509,612 filed on Jul. 20, 2011, the entire contents of each ofwhich are incorporated herein by reference thereto.

FIELD OF THE INVENTION

The present invention relates to a proteolytic extract obtained frombromelain for the treatment of connective tissue diseases. Inparticular, the present invention relates to a pharmaceuticalcomposition comprising a proteolytic extract obtained from bromelain forthe treatment of diseases such as Dupuytren's disease and Peyronie'sdisease.

BACKGROUND OF THE INVENTION

Collagen is the main component of the connective tissue and it is mostlyfound in fibrous tissues such as tendon, ligament and skin. Numerousdiseases and conditions are associated with excess collagen deposition,the most common are Dupuytren's disease and Peronei's disease.

Dupuytren's disease (DD) is a connective tissue disorder of abnormalcollagen production and deposition in the hand that is commonlycharacterized by contracture of metacarpophalangeal joints (MCPJs) andproximal interphalangeal joints (PIPJs) in the ring and little fingers.Fibroblast proliferation and differentiation into myofibroblasts withexcess collagen deposition at the level of the palmar fascia causenodule and fibrotic cord formation in the palm and/or digits. Thefibrotic cords or nodules can be of varying thicknesses, from 1millimeter in diameter for the fibrotic cords to nearly 10 millimetersin diameter for the fibrotic nodules. As the disease progresses, cordsbegin to contract, causing finger flexion-deformities (flexioncontractures) which interfere and decrease hand function.

The prevalence of DD increases with age and males are more oftenaffected. Genetic susceptibility, smoking, alcohol, diabetes mellitus,epilepsy and repetitive manual work are thought to be common riskfactors for DD. The severity and progress of DD can be classified by theaffected degree of digital flexion-contracture.

Surgical fasciectomy is currently the most widely available treatmentfor DD which provides positive, though temporary outcomes for mostpatients. However, surgical fasciectomy usually involves common surgicalcomplications (e.g., infection, hematoma, tissue loss) as well asspecific complications such as digital nerve damage, loss of fingers,skin flap loss, wound healing problems and postoperative stiffness. Inaddition, fasciectomy involves a long recovery and does not offer adefinitive cure as DD has an extremely high recurrence rate. Minimallyinvasive procedures using needles or thin blades have been tried; suchprocedures though cause less complications, increase the recurrencerate. Non surgical interventions have also been developed and includeradiotherapy, ultrasound, injection of vitamin A, vitamin E, steroidsand interferon-γ.

In vitro studies have demonstrated the ability of collagenase todecrease the tensile modulus and the force needed to rupture Dupuytren'scord tissue, indicating that collagenase may be effective in enzymaticfasciotomy. Clinical studies have recently demonstrated that treatmentwith Clostridium histolyticum collagenase released DD contractures andimproved the range of motion in affected joints. An 8-year follow-up ofcollagenase injection in patients with DD showed that the MCPJcontracture was less severe following the recurrence of the disease,when compared to the initial contracture before applying the collagenasetreatment. It has also been shown that Type-III collagen, which isusually absent from normal adult palmar fascia, is abundant in thetissue of patients with DD.

Peyronie's disease is a connective tissue disorder involving the growthof fibrous plaques rich in collagen in the soft tissue of the penisaffecting up to 10% of men. Specifically, the fibrous plaques are formedin the tunica albuginea, the thick sheath of tissue surrounding thecorpora cavernosa, cause abnormal curvature which is often associatedwith pain.

Surgery is the only approach to treating Peyronie's disease whichappears to have predictably repeatable efficacy. Surgery is usually onlyindicated in long-term cases where the disease is stabilized and thedeformity prevents intercourse and/or causes extreme pain. However,complications can develop from surgery, including a permanent shorteningof the penis.

Non-surgical approaches to Peyronie's disease treatment are alsoavailable, although they are all largely ineffective. Attempts todissolve the plaques by direct intra-lesional injections have beentried. Of the injection methodologies, those involving clostridialcollagenase appear to exhibit the most consistent efficacy, though stillquite limited in effect and duration. In addition, radiation therapy andlaser technology have been tried.

U.S. Pat. Nos. 5,589,171, 6,086,872 and Reissued U.S. Pat. No. RE39,941disclose methods of treating an individual suffering from Dupuytren'sdisease which methods comprise applying collagenase to a fibrousaffected palmar fascia.

U.S. Pat. No. 6,022,539 discloses methods of treating an individualsuffering from Peyronie's disease which methods comprise injectingcollagenase into a fibrous Peyronie's plaque in the penis of theindividual.

U.S. Pat. No. 6,353,028 discloses topical medicament which comprisescalcium channel blocker agents and carrier agents facilitatingtransdermal delivery of the calcium channel blocker for the treatment ofconnective tissue disorders: Peyronie's disease, Dupuytren's disease andLedderhose Fibrosis.

U.S. Patent Application Publication No. 2008/0206228 discloses amedicament containing hyaluronic acid or derivatives thereof inassociation with collagenase for the treatment of various kinds ofwounds, burns, pressure sores, vascular ulcers, and diabetic foot ulcersas well as for the treatment of hypertrophic and keloid scars. Treatmentof Dupuytren's disease is explicitly disclosed.

International Patent Application Publication No. WO 2004/037183discloses methods and compositions for treatment of conditions involvingfibrosis, among which Peyronie's disease and Dupuytren's disease aredisclosed. The compositions comprise a phosphodiesterase (PDE)-4inhibitor, a PDE-5 inhibitor or a compound that elevates cGMP, to listsome.

Use of cell cycle inhibitors, including anti-microtubule agents,antimetabolites, alkylating agents, vinca alkaloids, PDE inhibitors,matrix metalloproteinase including collagenases, for treating acontracture such as Dupuytren's contracture or Peyronie's contracture isdisclosed in International Patent Application Publication No. WO2005/074913.

Nowhere in the background art is it disclosed or suggested thatproteolytic enzymes from plant sources are useful for treatingconnective tissue disorders involving excess collagen deposition.

Extracts derived from the stem of the pineapple plant (Ananas comosus)have been found to selectively remove devitalized tissue. Such extracts,also named bromelain, contain various proteolytic and hydrolyticenzymes.

International Patent Application Publication No. WO 2006/054309 to theapplicant of the present invention discloses a debriding compositionobtained from bromelain comprising most of the proteolytic enzymespresent in bromelain, the proteolytic enzymes having an averagemolecular weight of 23 kDa. WO 2006/054309 further discloses uses ofsaid debriding composition for debriding non-viable tissues.

There remains an unmet need for improved non-invasive methods fortreating connective tissue diseases involving excess collagendeposition.

SUMMARY OF THE INVENTION

The present invention provides a proteolytic extract obtained frombromelain for the treatment of connective tissue diseases. Particularly,the present invention provides a proteolytic extract obtained frombromelain for the treatment of connective tissue diseases which areassociated with excess of collagen deposition, including Dupuytren'sdisease and Peyronie's disease.

It is now disclosed for the first time that a proteolytic extractobtained from bromelain comprising one or more of the cysteine proteasespresent in bromelain, e.g., stem bromelain or ananain, is capable ofdegrading native, non-denatured collagen. Unexpectedly, injection of theproteolytic extract into a Dupuytren's cord resulted in rupture of thecord while maintaining the normal healthy connective tissue intact.

The present invention further discloses that the efficacy of theproteolytic extract to rupture or dissolve Dupuytren's cords is similarto or even higher than that of collagenase. However, while collagenasemay cause damage to non-diseased ligaments or tendons due to itsaffinity to the various types of collagen, the proteolytic extract ofthe present invention shows specificity to the diseased cords. Thus, theproteolytic extract of the present invention provides an improved andsafe medication for connective tissue diseases which involve excesscollagen deposition, particularly for the treatment of Dupuytren'sdisease and Peronei's disease.

Due to the fact that high concentrations of the proteolytic extract canbe prepared in small volumes, such small volumes can be injected intothe diseased fibrous cords or plaques, thus avoiding extravasation anddamage to surrounding tissues, simplifying the clinical procedure andhence increasing patient's compliance.

According to one aspect, the present invention provides a method oftreating a connective tissue disease comprising administering to asubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount of a proteolytic extractobtained from bromelain and a pharmaceutically acceptable carrier,wherein the proteolytic extract comprises at least one cysteine proteaseselected from the group consisting of stem bromelain and ananain, andwherein the connective tissue disease is associated with excess collagendeposition.

According to additional embodiments, the connective tissue disease isselected from the group consisting of Dupuytren's disease, Peyronie'sdisease, frozen shoulder, and Ledderhose disease. According to a certainembodiment, the connective tissue disease is Dupuytren's disease.According to another embodiment, the connective tissue disease isPeronei's disease.

According to one embodiment, the proteolytic extract comprises stembromelain and ananain. According to another embodiment, the proteolyticextract further comprises a cysteine protease precursor. According to afurther embodiment, the proteolytic extract further comprises a cysteineprotease fragment. According to an exemplary embodiment, the proteolyticextract comprises stem bromelain, ananain, and a cysteine proteaseprecursor.

According to further embodiments, the pharmaceutical composition furthercomprising an agent selected from the group consisting of an anestheticagent, antibacterial agent and an anti-inflammatory agent.

According to still further embodiments, the anesthetic agent is selectedfrom the group consisting of amethocaine (tetracaine), lignocaine(lidocaine), xylocaine, bupivacaine, prilocaine, ropivacaine,benzocaine, mepivocaine, cocaine and combinations thereof. Eachpossibility is a separate embodiment of the invention.

According to additional embodiments, the antibacterial agent is selectedfrom the group consisting of amanfadine hydrochloride, amanfadinesulfate, amikacin, amikacin sulfate, amoglycosides, amoxicillin,ampicillin, amsamycins, bacitracin, beta-lactams, candicidin,capreomycin, carbenicillin, cephalexin, cephaloridine, cephalothin,cefazolin, cephapirin, cephradine, cephaloglycin, chilomphenicols,chlorhexidine, chloshexidine gluconate, chlorhexidine hydrochloride,chloroxine, chlorquiraldol, chlortetracycline, chlortetracyclinehydrochloride, ciprofloxacin, circulin, clindamycin, clindamycinhydrochloride, clotrimazole, cloxacillin, demeclocycline,diclosxacillin, diiodohydroxyquin, doxycycline, ethambutol, ethambutolhydrochloride, erythromycin, erythromycin estolate, erhmycin stearate,farnesol, floxacillin, gentamicin, gentamicin sulfate, gramicidin,giseofulvin, haloprogin, haloquinol, hexachlorophene, iminocylcline,iodochlorhydroxyquin, kanamycin, kanamycin sulfate, lincomycin,lineomycin, lineomycin hydrochloride, macrolides, meclocycline,methacycline, methacycline hydrochloride, methenine, methenaminehippurate, methenamine mandelate, methicillin, metonidazole, miconazole,miconazole hydrochloride, minocycline, minocycline hydrochloride,mupirocin, nafcillin, neomycin, neomycin sulfate, netimicin, netilmicinsulfate, nitrofurazone, norfloxacin, nystatin, octopirox, oleandomycin,orcephalosporins, oxacillin, oxyteacline, oxytetracycline hydrochloride,parachlorometa xylenol, paromomycin, paromomycin sulfate, penicillins,penicillin G, penicillin V, pentamidine, pentamidine hydrochloride,phenethicillin, polymyxins, quinolones, streptomycin sulfate,tetracycline, tobramycin, tolnaftate, triclosan, trifampin, rifamycin,rolitetracycline, silver salts, spectinomycin, spiramycin, struptomycin,sulfonamide, tetracyclines, tetracycline, tobramycin, tobramycinsulfate, triclocarbon, triclosan, trimethoprim-sulfamethoxazole,tylosin, vancomycin, and yrothricin. Each possibility is a separateembodiment of the invention.

According to further embodiments, the anti-inflammatory agent isselected from the group consisting of non-steroidal anti-inflammatoryagents and steroidal anti-inflammatory agents.

According to yet further embodiments, the pharmaceutical compositionfurther comprises a component selected from the group consisting of astabilizing agent, an anti-oxidant, a preservative, a buffering agent, achelating agent, and a tonicity agent.

According to still further embodiments, the pharmaceutical compositionis formulated in a form selected from the group consisting of a solidformulation, a semi solid formulation, a liquid formulation and a foamformulation. According to a certain embodiment, the solid formulation isa powder. According to another embodiment, the liquid formulation is aninjectable solution of a pH of about 6 to about 7.

According to an exemplary embodiment, the pharmaceutical composition isadministered by injection into the diseased fibrous tissue. Thepharmaceutical composition can be injected as a single dose or inaliquots at two or more locations in the diseased fibrous tissue.

According to another aspect, the present invention provides apharmaceutical composition comprising a proteolytic extract for use inthe treatment of a connective tissue disease, wherein the proteolyticextract comprises at least one cysteine protease selected from the groupconsisting of stem bromelain and ananain, and wherein the connectivetissue disease is associated with excess collagen deposition.

These and other embodiments of the present invention will be betterunderstood in relation to the figures, description, examples, and claimsthat follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the collagenolytic activity of two batches ofthe proteolytic extract. Increasing concentrations of the proteolyticextracts (designated MD2 H-05-27 and MD5 H-10-46) were incubated for 20minutes in the presence of fluorescently labeled collagen type IV. Atthe end of the incubation fluorescence was measured. Results arepresented in relative fluorescence units (RFU).

FIG. 2 is a graph showing the collagenolytic activity of the proteolyticextract on collagen type I and type IV. The proteolytic extract wasincubated in the presence of fluorescently labeled collagen type I orcollagen type IV for various time periods. At the end of the incubationfluorescence was measured. Results are presented in relativefluorescence units (RFU).

FIG. 3 is a graph showing the collagenolytic activity of the proteolyticextract as compared to that of collagenase. Clostridium histoliticumcollagenase was incubated with either fluorescently labeled collagentype IV or fluorescently labeled gelatin and fluorescence was measuredat the end of 20 minutes of incubation. The proteolytic extract wasincubated with fluorescently labeled collagen type IV for 20 minutes andthereafter fluorescence was measured.

FIG. 4 is a graph showing the gelatinase activity of the proteolyticextract. Increasing concentrations of two batches of the proteolyticextract (designated J-01-19 and J-14-45) were incubated for 20 minutesin the presence of fluorescently labeled gelatin and thereafterfluorescence was measured.

FIGS. 5A-C are photographs showing surgical excision-removal of theDupuytren's cord from a patient. FIG. 5A is a photograph showing theabnormal contracture of the ring finger in a patient with Dupuytren'sdisease. FIG. 5B is a photograph showing the surgical removal of thepathological cord. FIG. 5C is a photograph showing the cord afterremoval from the palmar bed.

FIG. 6 is a photograph showing the dissection of the Dupuytren's cord totwo.

FIG. 7 is a photograph showing the anchoring of the cord.

FIG. 8 is a photograph showing the injecting step of a solution into theDupuytren's cord.

FIG. 9 is a photograph showing the tensile stretching machine.

FIGS. 10A-B are photographs showing the cord before and after tensileforce application.

FIG. 10A shows the cord before tensile force application. FIG. 10B showsthe cord after tensile force application and before cord rupture.

FIG. 11 shows the effect of saline on Dupuytren's cord elongation as afunction of tensile strength application. Dupuytren's cord was injectedwith saline and incubated in saline for 24 hours. Thereafter, the cordwas subjected to tensile stress, and cord elongation and rupture wasevaluated.

FIG. 12 shows the effect of the proteolytic extract on Dupuytren's cordelongation as a function of tensile strength application. Dupuytren'scord was injected with the proteolytic extract and incubated in thepresence of the proteolytic extract for 24 hours. Thereafter, the cordwas subjected to tensile stress, and cord elongation and rupture wasevaluated.

FIG. 13 shows the effect of a single injection of the proteolyticextract on Dupuytren's cord elongation as a function of tensile strengthapplication. Dupuytren's cords were injected with the proteolyticextract and incubated in saline for 24 hours. Thereafter, the cords weresubjected to tensile stress, and cord elongation and rupture wereevaluated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating connective tissuediseases involving excess collagen deposition comprising administeringto a subject in need of such treatment a proteolytic extract obtainedfrom bromelain.

A debriding composition obtained from bromelain (also termed Debrase®)was first disclosed in WO 2006/054309 to the applicant of the presentinvention, the content of which is incorporated by reference as if fullyset forth herein. The debriding composition disclosed in WO 2006/054309comprises cysteine proteases such as stem bromelain and ananain. WO2006/054309 further discloses that the debriding composition debridedburned skin, i.e., devitalized tissue, more efficiently than bromelain.However, the debriding composition was found to be inactive in debridinghealthy or vital skin or dermis (see, for example, Singer et al., 2010,J. Burn Care Res. 31: 304-309). The debriding composition was thereforeshown to be active against devitalized tissues, not against viabletissues.

Unexpectedly, the present invention discloses that a proteolytic extractobtained from bromelain exerted collgenolytic activity in vitro and wascapable of dissolving the palmar fibrotic cords obtained from subjectssuffering from Dupuytren's disease. As the proteolytic extract of thepresent invention does not degrade healthy connective tissue, thepresent invention thus provides safe and efficient enzymatic medicamentfor dissolving fibrous tissue rich in collagen, specifically in subjectssuffering from Dupuytren's disease or Peronei's disease.

The terms “proteolytic extract obtained from bromelain” and “proteolyticextract” are used interchangeably throughout the specification andclaims and refer to an enzymatic preparation partially purified frombromelain.

The term “bromelain” refers to any of a number of presently commerciallyavailable bromelain powder preparations. Examples of manufacturers ofbromelain include, but are not limited to, Sigma and ChallengeBioproducts Co. Ltd., Taiwan. Bromelain is prepared from the stem ofpineapple plant. A typical procedure to obtain bromelain is as follows:the juice from the stem of pineapple plant is first adjusted to a pH ofabout 3 or 4 with phosphoric acid, and sodium hydride or sodiumsulfhydride is added to protect against sulfhydryl oxidation. The inertmaterial is precipitated at about 30% acetone and, after filtration, theclarified fluid is precipitated with 70% acetone. This precipitate iscollected by centrifugation and either redissolved in water containingsodium hydride or sodium sulfhydride which has been acidified withphosphoric acid and reprecipitated, or dried in a vacuum oven directly.If the material is reprecipitated, 70% acetone is utilized. The driedmaterial from either process is suitable as a starting material toobtain the debriding composition of the present invention.

The proteolytic extract of the present invention can comprise one ormore of the cysteine proteases present in bromelain. According to anexemplary embodiment, the proteolytic extract (also termed Debrase® orNexobrid®) comprises the cysteine proteases stem bromelain (EC3.4.22.32) and ananain (EC 3.4.22.31). The proteolytic extract canfurther comprise one or more of the cysteine protease precursors ofbromelain such as, for example, ananain (EC 3.4.22.31) precursor, fruitbromelain (EC 3.4.22.33) precursor, and stem bromelain (EC 3.4.22.31)precursor. The proteolytic extract can further comprise cysteineprotease fragments (see, for example, WO 2006/054309), a jacalin-likelectin (see, for example, Raval et al., Glycobiology, 2004, 14(12):1247-1263), and/or bromelain inhibitors.

The proteolytic extract can be prepared by a method comprising thefollowing steps:

-   -   (a) suspending bromelain with an acidic solution optionally        comprising an anti-oxidant, the acidic solution having a pH in        the range from about 2.4 to about 4;    -   (b) adjusting the suspension of (a) to a pH in the range from        about 2.4 to about 4;    -   (c) adding a filter aid to the suspension of (b);    -   (d) filtering the suspension of (c) to remove insoluble        components;    -   (e) adding to the filtered solution of (d) ammonium sulfate salt        to yield saturation of ammonium sulfate in the range from about        40% to about 50%;    -   (f) adjusting the suspension of (e) to a pH from about 2.5 to        about 4;    -   (g) incubating the suspension of (f) at 3° C.-10° C.;    -   (h) centrifuging the suspension of (g) to yield an ammonium        sulfate precipitate;    -   (i) dissolving the ammonium sulfate precipitate in an acidic        solution optionally comprising an anti-oxidant having a pH in        the range from about 2.4 to about 4;    -   (j) filtering the solution of (i) through a 10 kDa ultra-filter;        and    -   (k) lyophilizing the retained solution of (j).

According to some embodiments, suspending bromelain can be performed inany acidic solution having a pH between about 2.4 to 4. Examples ofacidic solutions or buffers that can be used according to the presentinvention include, but are not limited to, acetic acid in water, acetatebuffer and acetate buffer containing 1% thioglycolic acid, pH 2.4-4.According to certain exemplary embodiments, the acidic solution isselected from the buffers and solutions disclosed in U.S. Pat. Nos.5,830,739 and 4,197,291, the content of which is incorporated byreference as if fully set forth herein.

The acidic solution can optionally comprise an anti-oxidant. Examples ofanti-oxidants include, but are not limited to, ascorbic acid,dihydroquinon, butylated hydroxytoluene and dithiothreitol. Theanti-oxidant can be added at a concentration of about 0.5% to about 2%,preferably at 1%.

The acidic solution can further comprise a wetting agent. Examples ofwetting agents include, but are not limited to, n-octanol.

The pH of the acidic solution, which optionally comprises ananti-oxidant, can be in the range from about 2.4 to about 4. Accordingto a certain preferred embodiment, the pH of the acidic solution, whichoptionally comprises an anti-oxidant, ranges from about 2.4 to about2.6.

According to additional embodiments, a filter aid is added to thesuspension of (a). According to one embodiment, the filter aid comprisessilica. Preferably, the filter aid is natural diatomite that is calcinedso that faster flow rates are achieved.

Precipitating the desired proteins is performed by adding to thefiltered solution of step (d) ammonium sulfate salt. Ammonium sulfatesalt can be added to yield saturation of the ammonium sulfate at a rangeof between about 40% to about 50%. Preferably, ammonium sulfate salt canbe added to yield 40% saturation of ammonium sulfate.

The suspension of step (f) is then incubated at a temperature between 3°C. to 10° C. Preferably, the suspension of step (f) is incubated for atleast 10 hours at temperatures between 3° C. to 10° C. More preferably,the suspension of step (f) is incubated for 12-24 hours at 4° C.

At the end of the incubation, the suspension of step (g) is centrifugedto precipitate the desired proteins, i.e., the proteolytic enzymes. Theprecipitate is then dissolved in acidic solution optionally comprisingan anti-oxidant. According to an exemplary embodiment, the suspension isincubated for at least 10 hours at 4° C.

The solution of step (i) is subjected to a step of filtering to retainproteolytic enzymes having molecular weights in excess of about 10 kDa.According to a preferred embodiment, the solution of step (i) isfiltered through a membrane filter having a molecular weight cut off ofabout 10 kDa.

The proteolytic extract can be lyophilized after filtration, can bewashed with distilled water and then lyophilized or can be filtered andthen lyophilized. According to a currently preferred embodiment, theproteolytic extract is filtered through a filter membrane having a poresize of at least about 0.5 μm to obtain a sterile solution, which isthen lyophilized and stored. Preferably, the proteolytic extract isstored as a lyophilized powder as its stability is prolonged in theabsence of moisture. Prior to use, the proteolytic extract is dissolvedin a solution so as to obtain a solution with a pH of about 6 to about7.

According to an exemplary embodiment, the proteolytic extract can beprepared by the method comprising the following steps:

-   -   (a) suspending bromelain with 0.3 M acetic acid comprising 1%        ascorbic acid and n-octanol having a pH from about 2.4 to about        2.6;    -   (b) adjusting the suspension of (a) to a pH in the range from        about 2.5 to about 3.5;    -   (c) adding a filter aid comprising silica to the suspension of        (b);    -   (d) filtering the suspension of (c) through a filter press to        remove insoluble components;    -   (e) adding to the filtered solution of (d) ammonium sulfate salt        (285 g/L) to yield 40% saturation of ammonium sulfate;    -   (f) adjusting the suspension of (e) to a pH from about 2.5 to        about 3.5;    -   (g) incubating the suspension of (f) for approximately 12-24        hours at 4° C.;    -   (h) centrifuging the suspension of (g) to yield an ammonium        sulfate precipitate;    -   (i) dissolving the ammonium sulfate precipitate in 0.3 M acetic        acid comprising 1% ascorbic acid having a pH from about 2.4 to        about 2.6;    -   (j) filtering the solution of (i) through a 10 kDa ultra-filter;    -   (k) filtering the retained solution of (j) to yield a sterile        solution; and    -   (l) lyophilizing the filtered solution of (k).

The term “Dupuytren's disease” and “DD” are interchangeably used hereinand refer to a disease where the fingers cannot fully extend and areusually flexed towards the palm of the hand. Specifically, Dupuytren'sdisease begins with the formation of fibromatous nodules in the palmarfascia, usually in the ulnar side. The nodules progress and form afibrous band or cord lying from the palm to the fingers. Eventually thisleads to permanent finger flexion-contractures. The ring finger is mostcommonly affected, followed by the little finger.

The terms “Dupuytren's cord” and “the diseased cord” are used hereininterchangeably and refer to the bands of fascial fibers that runlongitudinally bellow the palmar skin. These bands lead to contracturesof the overlay skin and distal digits that are attached to the bands andeventually progresses to permanent flexion-contracture of the affecteddigits. Typically, the Dupuytren's cord comprises high numbers offibroblasts, increased deposition of extracellular matrix (ECM)proteins, particularly collagen, and myofibroblasts.

It is to be understood that the proteolytic extract of the invention isuseful for treating individuals having other diseases associated withexcess collagen deposition. Other fibrous tissue malformations andabnormalities which involve collagen deposition include Peyronie'sdisease, Ledderhose Fibrosis, and fibrosis of joint-capsules, tendonsand ligaments sheaths. Thus, the proteolytic extract can also be usefulfor treating frozen shoulder (adhesive capsulitis). These connectivetissue diseases involving excess collagen deposition or fibrous tissuemalformations are not associated with wounds or burns.

As used herein, the terms “treating” or “treatment” refer toamelioration or elimination of at least one or more of the symptomsassociated with a connective tissue disease. For example, symptomsassociated with Dupuytren's disease include joint contracture, decreaserange of motion of the joints, to list some. Symptoms of Peyronie'sdisease include, for example, pain, abnormal curvature, and erectiledysfunction.

The term “therapeutically effective amount” of the proteolytic extractis that amount of the proteolytic extract which is sufficient to providea beneficial effect to the subject to which the composition isadministered.

The term “about” when refers to a pH of a solution or suspension ismeant to indicate that 0.5 pH units above or below the indicated pH arewithin the scope of the present invention.

The pharmaceutical composition of the present invention comprises theproteolytic extract and a pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans.

The term “carrier” refers to a diluent, excipient, or vehicle with whichthe proteolytic extract is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents. Salinesolutions, aqueous NaCl/CaCl2 solution, aqueous dextrose, glycerolsolutions and albumin solutions can be employed as liquid carriers,particularly for injectable solutions. Water can also be used as acarrier when the pharmaceutical composition is administeredintravenously;

The pharmaceutical composition can further comprise stabilizing agentssuch as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gumacacia, calcium phosphate, alginates, tragacanth, calcium silicate,polyvinylpyrrolidone and cellulose. The composition can additionallyinclude lubricating agents, such as, magnesium stearate and mineral oil;wetting agents; emulsifying and suspending agents; preservatives such asThimerosal, benzyl alcohol, parabens, methyl- or propylhydroxybenzoates;anti-oxidants such as ascorbic acid, dihydroquinon, butylatedhydroxytoluene and dithiothreitol; and buffering agents such asmonobasic sodium phosphate, dibasic sodium phosphate, sodium benzoate,potassium benzoate, sodium citrate, sodium acetate, and sodium tartrate;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose.

The pharmaceutical composition can further comprise an anesthetic agent.

Anesthetic agents include, but are not limited to, amethocaine(tetracaine), lignocaine (lidocaine), xylocaine, bupivacaine,prilocaine, ropivacaine, benzocaine, mepivocaine, cocaine andcombinations thereof.

The pharmaceutical composition can further comprise an antibacterialagent.

Antibacterial agents include, but are not limited to, amanfadinehydrochloride, amanfadine sulfate, amikacin, amikacin sulfate,amoglycosides, amoxicillin, ampicillin, amsamycins, bacitracin,beta-lactams, candici din, capreomycin, carbenicillin, cephalexin,cephaloridine, cephalothin, cefazolin, cephapirin, cephradine,cephaloglycin, chilomphenicols, chlorhexidine, chloshexidine gluconate,chlorhexidine hydrochloride, chloroxine, chlorquiraldol,chlortetracycline, chlortetracycline hydrochloride, ciprofloxacin,circulin, clindamycin, clindamycin hydrochloride, clotrimazole,cloxacillin, demeclocycline, diclosxacillin, diiodohydroxyquin,doxycycline, ethambutol, ethambutol hydrochloride, erythromycin,erythromycin estolate, erhmycin stearate, farnesol, floxacillin,gentamicin, gentamicin sulfate, gramicidin, giseofulvin, haloprogin,haloquinol, hexachlorophene, iminocylcline, iodochlorhydroxyquin,kanamycin, kanamycin sulfate, lincomycin, lineomycin, lineomycinhydrochloride, macrolides, meclocycline, methacycline, methacyclinehydrochloride, methenine, methenamine hippurate, methenamine mandelate,methicillin, metonidazole, miconazole, miconazole hydrochloride,minocycline, minocycline hydrochloride, mupirocin, nafcillin, neomycin,neomycin sulfate, netimicin, netilmicin sulfate, nitrofurazone,norfloxacin, nystatin, octopirox, oleandomycin, orcephalosporins,oxacillin, oxyteacline, oxytetracycline hydrochloride, parachlorometaxylenol, paromomycin, paromomycin sulfate, penicillins, penicillin G,penicillin V, pentamidine, pentamidine hydrochloride, phenethicillin,polymyxins, quinolones, streptomycin sulfate, tetracycline, tobramycin,tolnaftate, triclosan, trifampin, rifamycin, rolitetracycline, silversalts, spectinomycin, spiramycin, struptomycin, sulfonamide,tetracyclines, tetracycline, tobramycin, tobramycin sulfate,triclocarbon, triclosan, trimethoprim-sulfamethoxazole, tylosin,vancomycin, and yrothricin.

According to yet another embodiment, the pharmaceutical composition canfurther comprise an anti-inflammatory agent.

The anti-inflammatory agent can be non-steroidal, steroidal, or acombination thereof. Non limiting examples of non-steroidalanti-inflammatory agents include, oxicams, such as piroxicam, isoxicam,tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid,benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal;acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin,sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin,acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, andketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic,niflumic, and tolfenamic acids; propionic acid derivatives, such asibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen,fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen,miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic;pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone,azapropazone, and trimethazone. Extracts of these non-steroidalanti-inflammatory agents may also be employed.

Non-limiting examples of steroidal anti-inflammatory drugs include,corticosteroids such as hydrocortisone, hydroxyl-triamcinolone,alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasonedipropionates, clobetasol valerate, desonide, desoxymethasone,desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasonediacetate, diflucortolone valerate, fluadrenolone, flucloroloneacetonide, fludrocortisone, flumethasone pivalate, fluosinoloneacetonide, fluocinonide, flucortine butylesters, fluocortolone,fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide,hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone,triamcinolone acetonide, cortisone, cortodoxone, flucetonide,fludrocorisone, difluorosone diacetate, fluradrenolone, fludrocortisone,diflurosone diacetate, fluradrenolone acetonide, medrysone, amcinafel,amcinafide, betamethasone and the balance of its esters,chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone,dichlorisone, diflurprednate, flucloronide, flunisolide,fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate,hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate,triamcinolone, and extracts thereof.

The pharmaceutical composition can be formulated as a dry or lyophilizedformulation, semi solid formulation, liquid formulation or a foamformulation. Thus, the pharmaceutical composition can be formulated inthe form of a powder, solution, suspension, emulsion, gel, spray, or apatch.

The pharmaceutical composition can be administered into the affectedsite topically, subcutaneously, intracutaneously, or intramuscularly.

According to a certain embodiment, the pharmaceutical composition isadministered by injection. According to an exemplary embodiment, thepharmaceutical composition is injected directly into the diseasedfibrous nodules or cord or into the fibrous plaque. Alternatively, thepharmaceutical composition is implanted into a surgical incision.Sterile injectable preparations may be formulated as aqueous solutionsor oleaginous suspensions as known in the art.

For topical use on the skin the pharmaceutical composition can beformulated in the form of an ointment, cream, lotion, paste, spray, oraerosol. Examples of suitable vehicles include, but are not limited to,petrolatum, aquaphor, neobase, propylene glycol, glycerin and the like.Combinations of two or more of these vehicles can also be used.

The pharmaceutical composition may be formulated as controlled orsustained release formulations allowing for extended release of theactive components over a predetermined time period. In a certainembodiment, the pharmaceutical composition is administered incombination with a biodegradable, biocompatible polymeric implant, whichreleases the proteolytic extract over a controlled period of time at aselected site. Examples of polymeric materials include polyanhydrides,polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinylacetate, copolymers and blends thereof (See, Medical applications ofcontrolled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton,Fla.). Alternatively, the pharmaceutical composition is appliedtopically as a gel. Examples of polymeric materials that can be used arepolysaccharides, particularly cellulose derivatives such as, forexample, hydroxypropyl cellulose, carboxymethyl cellulose, andhydroxyethyl cellulose, chitin, chitosan, and alginates. The gelformulation would allow for extended release of the active componentsover a predetermined period of time.

The pharmaceutical composition can be formulated as foam. Gaspropellants are used to generate and administer a foamable compositionas foam. Examples of suitable gas propellants include volatilehydrocarbons such as butane, propane, isobutane or mixtures thereof, andfluorocarbon gases. The composition may be aqueous, oil-in-wateremulsion or water-in-oil emulsion, further comprising a stabilizingagent. The stabilizing agent increases the viscosity of the composition,can contribute to the composition stability, and/or slows foam collapserate. Examples of stabilizing agents include, but are not limited to,naturally-occurring polymeric materials (e.g., alginate, albumin,carrageenan, xanthan gum, starch), semi-synthetic polymeric materialssuch as cellulose ethers (e.g. hydroxyethyl cellulose, methyl cellulose,carboxymethyl cellulose, hydroxy propylmethyl cellulose), and syntheticpolymeric materials (e.g., polyvinyl alcohol, carboxyvinyl polymers, andpolyvinylpyrrolidone).

Techniques for formulation and administration of drugs can be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

The pharmaceutical composition can be administered as a single dose, orin aliquots at two or more locations in the affected fibrous tissue. Theamount of the proteolytic extract to be administered is an effectiveamount which softens and/or ruptures the plaque. An effective amount ofthe proteolytic extract can range from about 0.2 mg/day to about 40mg/day. In a certain embodiment, the pharmaceutical composition isadministered in two or more aliquots, each comprising about 0.5-1.5 mg,optionally in 0.2-0.5 ml of solution or suspension.

In certain embodiments, the organ into which the pharmaceuticalcomposition comprising the proteolytic extract is administered isimmobilized for several hours, e.g., 2 to 12 hours.

Each possibility disclosed throughout the specification is a separateembodiment of the invention.

The following examples are presented to provide a more completeunderstanding of the invention. The specific techniques, conditions,materials, proportions and reported data set forth to illustrate theprinciples of the invention are exemplary and should not be construed aslimiting the scope of the invention.

EXAMPLE 1 The Collagenolytic Activity of the Proteolytic Extract

The proteolytic extract was obtained from bromelain as described in WO2006/054309.

The ability of two batches of Debrase to degrade collagen type IV wasfirst determined. The assay was based on EnzChek®Gelatinase/colllagenase Assay Kit (Invitrogen) which contained DQcollagen type IV™ labeled with fluorescein as a substrate. Thissubstrate is known to be efficiently digested by collagenases to yieldhighly fluorescent peptides. The increase in fluorescence isproportional to the proteolytic activity.

To each well, a Debrase reaction buffer (0.15 M Tris-HCl and 10 mM EDTA,pH 7.6) was added in order to obtain a final volume of 100 μL. Ten μL of0.5 μg/μl of DQ collagen type IV™ solution were then added to the wells.Thereafter, different volumes (10-80 μL) of freshly prepared Debrase ata concentration of 0.225-1 ng/μL were added to the wells in Debrasebuffer to achieve concentrations of 1.5-20 ng/well. Debrase buffer wasused as a negative control. Reaction plate was incubated at roomtemperature for 20 minutes. To stop the reaction, 20 μL of stop reactionsolution (0.324 mM iodoacetic acid in Debrase buffer) were added.Fluorescence intensity was measured by a fluorescence micro-plate reader(Analyst AD, LJL) equipped with standard fluorescein filters. Backgroundfluorescence from wells incubated in the absence of enzyme wassubtracted.

FIG. 1 shows the collagenolytic activity of two batches of Debrase. Asshown in the figure, the two batches of Debrase exerted similarcollagenolytic activity indicating that the experimental procedure forobtaining the proteolytic extract of the present invention yieldsconsistent enzyme preparations.

Next, the ability of the proteolytic extract to degrade collagen types Iand IV was determined. For that end, DQ collagen type IV™ and DQcollagen type I™ labeled with fluorescein were used as substrates. Theassay was performed as described herein above and continued for the timeperiods as indicated in FIG. 2. The reaction was stopped by the additionof 20 μL stop reaction solution (0.324 mM iodoacetic acid in Debrasebuffer) and the fluorescence intensity was measured as described hereinabove.

Collagenase purified from Clostridium histolyticum served as a positivecontrol with predefined activity (one unit was defined as the amount ofenzyme required to liberate 1 micromole of E-leucine equivalents fromcollagen in 5 hours at 37° C., pH 7.5).

For the assay with Clostridium histolyticum collagenase, a reactionbuffer for collagenase (0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, 0.2 mMsodium azide, pH 7.6) was added to obtain a final volume of 100 μL pereach well. Then, different volumes (10-80 μL) of Clostridiumcollagenase, 0.4-1 mU/μL, in Clostridium collagenase buffer where addedto reference wells to reach concentrations ranging from 5 to 80 mU/well.To stop the Clostridium collagenase reaction—20 μL of 2 mg/ml1,10-phenanthroline in collagenase buffer was added.

Data from Clostridium collagenase were used as a reference value permUnit. Data from the proteolytic extract (also named Debrase) sampleswere divided by the reference value to determine mU/ng Debrase.

FIG. 2 shows that the proteolytic extract degraded collagen type I andIV with a specific activity of 1.58 and 1.27 mU/ng, respectively.

Next, the activity of the proteolytic extract was compared toClostridium histolyticum collagenase activity against collagen. Theproteolytic activity of collagenase against gelatin was also measuredusing the EnzChek® Gelatinase/colllagenase Assay Kit (Invitrogen) and DQgelatin™ (Invitrogen) as a substrate.

FIG. 3 shows that the proteolytic extract exerted collagenolyticactivity against collagen type IV, which activity was higher than thatobtained by the commercially available collagenase against collagen.

FIG. 4 shows that the proteolytic extract exerted gelatinase activity.As shown in the figure, gelatinase activity of the proteolytic extractwas linear at a concentration range of 2-8 ng/well.

EXAMPLE 2 The Proteolytic Extract Facilitates Rupture of Dupuytren'sCord

Dupuytren's cords were obtained from patients undergoing fasciectomy(FIGS. 5A, 5B and 5C). Consent forms were signed by all subjects priorto surgery and the study was approved by a Helsinki committee. Theexperiment examined the capability of the proteolytic extract to performfasiectomy of the cord.

Tissue Preparation

The cords obtained from the patients were divided to two or threepieces, depending on their length (FIG. 6). The cords were connected bythe Krackov technique to a mechanical testing device via a prolene 1suture (Ethicon, Somerville, N.J.) (FIG. 7). One of the two cords wasinjected with the proteolytic extract (0.3-0.5 ml of the proteolyticextract according to the cord size) and the second, control cord, wasinjected with saline (FIG. 8). The cords of the proteolytic extractgroup were immersed in the proteolytic extract solution and the cords ofthe control group were immersed in saline. Both groups were incubated at37° for 24 hours.

Mechanical Testing

After 24 hours of incubation, all cords were connected to a mechanicaltensile stress testing device (Zwick 1445 testing system, Zwick Co.,Germany). Each cord was subjected to an increasing load until the cordor the connecting suture was ruptured. The device measured the appliedtensile force until rupture.

Histological Analysis

A sample of each specimen was obtained for histological analysis and fordetermination of the disease stage.

Statistical Analysis

The efficiency of the control group compared to the study group wastested using Fisher exact test.

Results

All the cords treated with the proteolytic extract (n=10) were rupturedfollowing stretching (FIGS. 10A and 10B). Some of the cords treated withthe proteolytic extract were almost completely ruptured, practicallydissolved prior to the mechanical test. All control cords (n=9) did notrupture following the tensile force application. As depicted in FIG. 11,all the control cords exhibited a similar stress elongation patternrepresented by a limited elongation with increasing stress of the corduntil ruptured when a very high load applied. In contrast, all the cordstreated with the proteolytic extract exhibited loss of the cord'stensile strength, culminating in rupture of the cord at a very lowstress (FIG. 12). The results demonstrated that low doses, i.e., 0.8mg/ml, of the proteolytic extract were capable of rupturing theDupuytren's diseases cords and that this effect was further demonstratedat higher doses of the proteolytic extract, i.e., up to 150 mg/ml.

In order to evaluate the effect of a single injection of the proteolyticextract on the stretching of Dupuytren's cord, the cords were injectedwith the proteolytic extract at three different sites along the cord andthen incubated in saline in the absence of the proteolytic extract for24 hours at 37° C.

FIG. 13 shows that injections of the proteolytic extract given at threesites of the Dupuytren's cords were capable of reducing the tensilestrength of the cords by a factor of 4-5 (see FIG. 13, test 1 a and 2showing cord rupture at a stress of 18 N and 15 N, respectively 13). Onecord (FIG. 13, test 1 b) underwent rupture at a stress of 0.4 N. Theseresults demonstrate the efficiency of the proteolytic extract todissolve Dupuytren's cords and clearly imply that the proteolyticextract of the present invention is a highly effective enzymaticmedication for Dupuytren's disease as well as for other connectivetissue diseases involving excess collagen deposition.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above. Rather the scope of the invention is defined bythe claims that follow.

1. A method of treating a connective tissue disease comprising injecting to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of a proteolytic extract obtained from bromelain and a pharmaceutically acceptable carrier, wherein the proteolytic extract comprises at least one cysteine protease selected from the group consisting of stem bromelain EC 3.4.22.32, and ananain EC 3.4.22.31, said proteolytic extract further comprises at least one cysteine protease precursor, wherein the connective tissue disease is associated with excess collagen deposition, and wherein the step of injecting is repeated two or more times.
 2. The method according to claim 1, wherein the connective tissue disease is selected from the group consisting of Dupuytren's disease, Peyronie's disease, frozen shoulder, and Ledderhose disease.
 3. The method according to claim 1, wherein the connective tissue disease is Dupuytren's disease.
 4. The method according to claim 1, wherein the connective tissue disease is Peyronie's disease.
 5. The method according to claim 1, wherein the proteolytic extract comprises stem bromelain EC 3.4.22.32, ananain EC 3.4.22.31, and at least one cysteine protease precursor.
 6. The method according to claim 1, wherein the pharmaceutical composition further comprises an agent selected from the group consisting of an anesthetic agent, an antibacterial agent, and an anti-inflammatory agent.
 7. The method according to claim 7, wherein the anesthetic agent is selected from the group consisting of amethocaine (tetracaine), lignocaine (lidocaine), xylocaine, bupivacaine, prilocaine, ropivacaine, benzocaine, mepivocaine, cocaine and combinations thereof.
 8. The method according to claim 7, wherein the antibacterial agent is selected from the group consisting of amanfadine hydrochloride, amanfadine sulfate, amikacin, amikacin sulfate, amoglycosides, amoxicillin, ampicillin, amsamycins, bacitracin, beta-lactams, candicidin, capreomycin, carbenicillin, cephalexin, cephaloridine, cephalothin, cefazolin, cephapirin, cephradine, cephaloglycin, chilomphenicols, chlorhexidine, chloshexidine gluconate, chlorhexidine hydrochloride, chloroxine, chlorquiraldol, chlortetracycline, chlortetracycline hydrochloride, ciprofloxacin, circulin, clindamycin, clindamycin hydrochloride, clotrimazole, cloxacillin, demeclocycline, diclosxacillin, diiodohydroxyquin, doxycycline, ethambutol, ethambutol hydrochloride, erythromycin, erythromycin estolate, erhmycin stearate, farnesol, floxacillin, gentamicin, gentamicin sulfate, gramicidin, giseofulvin, haloprogin, haloquinol, hexachlorophene, iminocyldine, iodochlorhydroxyquin, kanamycin, kanamycin sulfate, lincomycin, lineomycin, lineomycin hydrochloride, macrolides, meclocycline, methacycline, methacycline hydrochloride, methenine, methenamine hippurate, methenamine mandelate, methicillin, metonidazole, miconazole, miconazole hydrochloride, minocycline, minocycline hydrochloride, mupirocin, nafcillin, neomycin, neomycin sulfate, netimicin, netilmicin sulfate, nitrofurazone, norfloxacin, nystatin, octopirox, oleandomycin, orcephalosporins, oxacillin, oxyteacline, oxytetracycline hydrochloride, parachlorometa xylenol, paromomycin, paromomycin sulfate, penicillins, penicillin G, penicillin V, pentamidine, pentamidine hydrochloride, phenethicillin, polymyxins, quinolones, streptomycin sulfate, tetracycline, tobramycin, tolnaftate, triclosan, trifampin, rifamycin, rolitetracycline, silver salts, spectinomycin, spiramycin, struptomycin, sulfonamide, tetracyclines, tetracycline, tobramycin, tobramycin sulfate, triclocarbon, triclosan, trimethoprim-sulfamethoxazole, tylosin, vancomycin, and yrothricin.
 9. The method according to claim 7, wherein the anti-inflammatory agent is selected from the group consisting of non-steroidal anti-inflammatory agents, and steroidal anti-inflammatory agents.
 10. The method of claim 1, wherein the pharmaceutical composition further comprises a component selected from the group consisting of a stabilizing agent, an anti-oxidant, a preservative, a buffering agent, a chelating agent, and a tonicity agent.
 11. The method according to claim 1, wherein the pharmaceutical composition is formulated in a form selected from the group consisting of a solid formulation, a semi solid formulation, a liquid formulation, and a foam formulation.
 12. The method according to claim 11, wherein the solid formulation is a powder.
 13. The method according to claim 11, wherein the liquid formulation has a pH of about 6 to about
 7. 14. The method according to claim 1, wherein the pharmaceutical composition is injected into the diseased fibrous tissue.
 15. The method according to claim 5, wherein the proteolytic extract further comprises at least one cycteine protease fragment.
 16. The method according to claim 15, wherein the proteolytic extract comprises stem bromelain EC 3.4.22.32, ananain EC 3.4.22.31, at least one cysteine protease precursor, at least one cysteine protease fragment, and a lectin.
 17. The method according to claim 1, wherein the connective tissue disease associated with excess collagen deposition is selected from the group consisting of fibrous tissue malformations and fibrous tissue abnormalities.
 18. The method according to claim 17, wherein the fibrous tissue malformations and fibrous tissue abnormalities are selected from the group consisting of fibrosis of joint capsules, fibrosis of tendons, fibrosis of ligament sheaths, scars, hypertrophic scars, and keloid scars. 